Prion-free collagen and collagen-derived products and implants for multiple biomedical applications; methods of making thereof

ABSTRACT

The use of collagen as a biomedical implant raises safety issues towards viruses and prions. The physicochemical changes and the in vitro and in vivo biocompatibility of collagen treated with heat, and by formic acid (FA), trifluoroacetic acid (TFA), tetrafluoroethanol (TFE) and hexafluoroiso-propanol (HFIP) were investigated. FA and TFA resulted in extensive depurination of nucleic acids while HFIP and TFE did so to a lesser degree. The molecules of FA, and most importantly of TFA, remained within collagen. Although these two acids induced modification in the secondary structure of collagen, resistance to collagenase was not affected and, in vitro, cell growth was not impaired. Severe dehydrothermal treatment, for example 110° C. for 1-3 days under high vacuum, also succeeded in removing completely nucleic acids. Since this treatment also leads to slight cross-linking, it could be advantageously used to eliminate prion and to stabilize gelatin products. Finally, prolonged treatment with TFA provides a transparent collagen, which transparency is further enhanced by adding glycosaminoglycans or proteoglycans, particularly hyaluronic acid. All the above treatments could offer a safe and biocompatible collagen-derived material for diverse biomedical uses, by providing a virus or prion-free product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.provisional Application No. 60/010,794, filed Jan. 29, 1996, nowabandoned, and PCT Application No. PCT/CA97/00070, filed Jan. 29, 1997,now pending.

FIELD OF THE INVENTION

This invention relates to a method of eliminating prion for collagen orcollagen-derived products, particularly by dehydrothermal or chemicalinactivation.

BACKGROUND OF THE INVENTION

Among biological materials, collagen, particularly type I collagen, is amajor component of various connective tissues including bone. Thereplacement of human tissues with human- or animal-derived tissues suchas skin or bone grafts results in the improvement of the wound healingprocess because of the presence of collagen. Therefore, the applicationof collagen-derived products as biomaterials has tremendous impact inbiomedicine because of (i) the natural structure of these products as abiological support for cells and scaffold for tissue repair orregeneration, (ii) their biodegradability that obviates removal ofimplants, and (iii) their biocompatibility. Collagen has been used todesign biomaterials such as wound dressings, artificial dermis, bone ortendon substitutes, tissue engineered devices, and injectable materialsin plastic surgery ¹⁻⁵. One of the advantages of using animal collagen,particularly from bovine species, is the facility with which largequantities of pure type I or type I/III collagen can be produced.

The emergence of new viruses and the appearance of new infectivediseases require increased vigilance concerning the safety ofbiologicals, especially since the discovery of transmission of proteinsuch as the prion correlates with infectivity (i.e., bovine spongiformencephalopathy)⁶. The assessment of the safety of a biological producttowards prion is complicated by the lack of a test capable of detectingscrapie-like agents in the starting material. Collagen purified fromhuman sources could also be a vector for the human spongiformencephalopathies⁷ and, perhaps, viral diseases (known and unknown).

The scrapie agent is extremely resistant to heat and physicalinactivation⁸. Prolonged exposure to concentrated NaOH solutions andautoclaving at temperatures above 130° C. can be recommended for routineinactivation and disinfection of scrapie-like agents⁹. However, there isstill a debate over whether this procedure simply extends the incubationperiod¹⁰. Furthermore, only NaOH can be applied to collagen becauseautoclave destroys collagen.

Treatment of collagen by NaOH at similar concentrations and incubationperiods (as recommended) have been investigated towards the eliminationof other infectious agents such as bacterium or viruses (RNA or DNAviruses). In a basic environment, we have shown indirectly by agarosegel that DNA and RNA are not fragmented enough to implement eliminationof infectious disease and transmission.

In another study we have compared the effect of glutaraldehyde treatmentas usually used to crosslink collagen products (i.e., Yannas' skin;cardiac biological valves; vessel grafts; and other biologicalimplants). Two methods of treating collagen with glutaraldehyde solutionhave been tested. One is using water and the other diluted acetic acidas buffer to crosslink collagen. The latter condition is described inYannas' patent U.S. Pat. No. 4,060,081 on his artificial skin. Manyinvestigators claim that glutaraldehyde treatment of collagen andderived products (i.e., gelatin capsules) can sterilize the finalcrosslinked products. Using the two methods of glutaraldehyde treatment,our investigation (using agarose gel) shows clearly that DNA and RNAincorporated in our collagen are not completely broken down andsubsequently the risk of transmission of viruses and bacteria is mostprobable.

Other treatments such as 8M urea have been also recommended.Nevertheless, partial breakdown of DNA and RNA was observed. Thisbreakdown is partial enough to offer no warranty of virus-free products.

On the other hand, formic acid (FA), SDS, fluorinated alcohols, andtrifluoroacetic acid (TFA) have dramatic effects on the prion (PrP27-30)secondary and tertiary structures which correlates with the inactivationof scrapie infectivity¹¹⁻¹². There is no suggestion in the art thatcollagen will resist to those treatments.

As mentioned above, a combination of NaOH and autoclave is currentlyused to eliminate prion. We have shown that NaOH by itself cannotwarrant the breakdown of DNA or RNA. It is further known thatautoclaving at about 130° C. destroys collagen. Other procedures makinguse of severe dehydrothermal treatment are used for crosslinking polymercomponents. Dehydrothermal treatment also has for effect to sterilizethe products as well as crosslinking polymer components. Nobody hasinvestigated as to whether heat sterilization may remove prions whilepreserving the integrity of collagen-comprising products.

In view of the foregoing, there is a need for a method of making aprion-free collagen-comprising product wherein prion is eliminated whilecollagen is not substantially denatured beyond a desirable orunavoidable extent.

STATEMENT OF THE INVENTION

It is an object of the present invention to provide a method toeliminate prion from collagen-comprising products while preservingsubstantially the integrity of those products.

In a particular embodiment of the invention, such a method makes use ofa strong acid having a pH solution below about 2. In a preferredembodiment, the strong acid is pure trifluoroacetic acid or fluoroaceticacid (pH 1) applied directly on a lyophilized collagen-comprisingproduct by impregnation. The time of reaction may vary from about 1 to 5hours depending on the nature of the acid e.g. the more potent is theacid, lesser the time is necessary to eliminate prion without affectingthe integrity of the product.

In another embodiment of the invention, dehydrothermal treatmentsubstitutes for the chemical inactivations e.g. the strong acid. In thatparticular method, collagen a collagen-derivative such as gelatin issubmitted to temperature and time conditions which are sufficient toeliminate prion without affecting substantially the integrity ofcollagen beyond a desirable extent. In a preferred embodiment, thoseconditions are a temperature of 110° C. and a period of 1 to 3 days, ina dry atmosphere (under high vacuum).

In another embodiment of the invention, the two above-sterilizationmethods are combined. First, collagen is treated with TFA for a periodof time which is dependent on the desirability to convert collagen intogelatin. When a substantive conversion to gelatin is desirable, collagenmay be treated with TFA or an acid having an equivalent action, for aperiod of time which is higher than about 5 hours, preferably between 6to 12 hours. The collagen or collagen-derivative (e.g., for example,collagen, TFA-treated collagen or gelatin) are then submitted to adehydrothermal treatment, which has for effect to eliminate prion, ifany, and crosslink the product. The heat treatment has for dual effectto stabilize the product by crosslinking and eliminate prion.

It is another object of this invention to-provide products comprisingcollagen and collagen derivatives prepared by the acid and/orheat-inactivation above processes which have the advantage of achievinga safe prion-free collagen. Collagen as a starting material in theproduction of collagen products, collagen already/shaped as films,sponges, drug-delivery systems or wound dressings; collagen conjugatedto other acid-stable molecules, and collagen derivatives or fragments,are all examples of materials which be subject to inactivationprocesses.

This invention will be described hereinbelow by way of specific examplesand appended figures, which purpose is to illustrate and not to limitthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. FTIR spectra of collagen materials untreated (A) andtreated (for a 1 h exposure period) by formic acid (B); trifluoroaceticacid (C); trifluoroethanol (D), and hexafluoro-2-propanol (E).

FIG. 2. FTIR spectra of pure formic acid (FA); and trifluoroacetic acid(TFA).

FIGS. 3A-3D. FTIR spectra of collagen materials in the amide I regionbefore (a & b) and after (c & d) spectral deconvolution. Untreatedcollagen (full lines) were compared to collagen treated for 1 h (a & c)or 5 h (b & d) by formic acid (dash-dotted lines); trifluoroacetic acid(dashed lines); trifluoroethanol (dotted lines), andhexafluoro-2-propanol (dashed-double-dotted lines).

FIG. 4. Cell growth. Human dermal fibroblasts were cultivated onmultiwell plates in fluid medium contact with collagen sponges whichwere either untreated (open squares), or treated by exposure to FA(closed triangles), TFA (closed squares), TFE (closed circles), or HFIP(open circles). Cell growth was determined by counting cells as afunction of time. Means and standard deviations of the mean arepresented (N=18).

FIGS. 5A-5E. Observations of collagen sponges treated with FA (A, B &C); TFA (D), and TFE (E). Prior to implantation (A: Histologic section,x181; B: TEM, x19,200), the treatment of a porous collagen sponge by FAresults in a collapsed structure with collagen bundles (arrows) whichhave a periodicity (B). Subcutaneous implantations were performed inmice, and sequentially analyzed. Histologic sections of the 30 dayretrieved implants are presented (C, D & E: x90). At this period, cell(arrowheads) infiltration is present between the collagen bundles(arrows) of the implanted collagen materials. In B, note the depositionof extracellular matrix (*). In D, fatty tissue (f) was present withinthe collagen materials as well as inflammatory cells (i).

FIGS. 6A-6C. Average fragment length of nucleic acids contained withinbovine type I collagen and added following different chemical treatmentsof the collagen. Lanes in A contain collagen sponge plus 5 μg of yeastRNA; lanes in B contain collagen sponge plus 5 μg of calf thymus DNA;and lanes in C contain collagen sponge only. These samples were treatedwith the following chemicals: no chemical (lanes 1), formic acid (lanes2), trifluoroacetic acid (lanes 3), trifluoroethanol (lanes 4) andhexafluoro-2-propanol (lanes 5). The 1.5% agarose gel was stained withethidium bromide (1 μg/ml). The first and last lanes of this gel containHindIII lambda phage+HaeIII digested φX174 DNA molecular weightstandards, respectively.

FIG. 7. Water uptake of freeze-dried collagen sponges treated by FA orTFA for 1 hr exposure. Composite PEG-collagen materials were alsocompared before and after treatment with FA or TFA.

FIG. 8. Kinetic curves of radiolabeled growth factor (¹²⁵I-bFGF)absorbed in collagen and PEG-collagen sponges treated by FA or TFA for 1hr. Sponges were implanted for different periods and radioactivity wasmeasured as a function of time post-implantation.

FIG. 9. Optic densities of transparent collagen hydrogels and films.Various wavelengths were investigated towards a variety of collagenmaterials treated by TFA for 8 hrs.

DESCRIPTION OF THE INVENTION

Materials and Methods

Chemical Reagents

Formic acid (FA) (23.4N) was purchased from BDH Inc. (Ville St-Laurent,QC, Canada); trifluoroacetic acid (TFA) (free acid in ampuls) from SigmaChemical Co., (St-Louis, Mich., U.S.A.); 2,2,2-Tri-fluoroethanol (99%;TFE), and 1,1,1,3,3,3-Hexafluoro-2-propanol (99%; HFIP) from AldrichChemical Company (Milwaukee, Wis., U.S.A.).

Specimen Preparations

Collagen was extracted from adult bovine hide by acetic acid dispersionand purified by NaCl salt precipitation to obtain a dispersion ofinsoluble collagen fibril bundles¹³⁻¹⁴. The periodicity of thesecollagen fibrils was mostly preserved as previously reported¹⁴. Collagensponges were obtained by freeze-drying a 1% collagen dispersion (w/vcollagen to water) as previously described¹⁴. Sponges were then exposedto pure FA (pH: 1), TPA (pH: 1), TFE (pH: 5), and HPIP (pH: 4.5-5) fordifferent periods, according to the methodology described by Safar etal.¹¹. Vacuum dried specimens were then rehydrated in distilled anddeionized water with extensive washing for 24 h at room temperature.During this procedure, the pH resumed to a range of the initial water pHat 5-6 in less than 1 h and remained stable. Most samples were thenair-dried to be processed for analyses. For the cell culture and animalstudies, sponges were manipulated in sterile conditions.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra were recorded with a Nicolet Magna-550 Fourier transforminfrared spectrometer equipped with a MCT/A detector and a germaniumcoated KBr beamsplitter. One hundred scans were routinely acquired withan optical retardation of 0.5 cm, triangularly apodized and Fouriertransform to yield a 2 cm⁻¹ resolution. The attenuated total reflectancemode was used to record the infrared spectra of collagen sponges with aSplit Pea attachment equipped with a Si hemispherical, 3 mm diameterinternal reflection element. Fourier deconvolution of the amide Ispectral region was done using a narrowing factor of 7.5 and anapodization filter of 0.14 as described¹⁵. These parameters were used tominimize side lobes in the 1720-1750 cm⁻¹ region where no collagen bandwas observed. Measurements were made in triplicate on different collagenbatches for each treatment, including control.

Differential Scanning Calorimetry (DSC)

The denaturation temperatures were measured on a Perkin Elmer DSC 7differential scanning calorimeter. To facilitate the measurements, a 3%(v/w) collagen dispersion was produced and then air-dried to obtain acompact film. Specimens were introduced in a clean crimpable aluminumpan and about 15 ml of distilled water was added. A control pan filledwith the same volume of distilled water was set in the reference port ofthe instrument. The DSC was filled with liquid nitrogen and purged withhelium. Analyses were performed between −50° C. to 90° C. at the heatingrate of 10° C./min. Denaturation temperatures were determined bymeasuring the temperature reaching the highest point on the denaturationpeak. Measurements were made in duplicate and the denaturationtemperature values have a ±3° C. accuracy range.

Collagenase Assay

For the collagenase assays, dried collagen sponges were immersed incollagenase (250 units of collagenase per mg of collagen; type IA fromClostridium histolyticum, Sigma) neutral solution (pH 7.5) containingbuffer A (25 mM Tris buffer and 10 mM CaCl₂). Specimens were incubatedat 37° C. and observed at 5 min intervals during the incubation. Theincubation period that resulted in complete disappearance (i.e., allfragments) of collagen sponges was considered to be related with theresistance of the materials to enzyme degradation. In addition,hydroxyproline content was measured after a one-hour period ofcollagenase digestion according to a method previously described¹⁶.Triplicate samples were assayed and a student's t-test was used forcomparison with a level of significance set at ≦0.05.

Fibroblast Cell Cultures

Cell culture investigations were performed with human foreskinfibroblasts, seeded at a low cell density of 1×10³ cells/cm², on thebottom of wells. Collagen materials were introduced in cell cultureinserts (3.0 μm polyethylene terephthalate porous filter, from BectonDickinson Labware). Cells were cultivated in Dulbecco's Modified EagleMedium (Sigma) supplemented with 5% fetal bovine serum (GIBCO/BRL) andantibiotics, at 37° C. under humid atmosphere in 5% CO₂. At 24, 72 h and7 days, cell counts were determined directly in wells using a supravitalDNA stain (Hoechst 33342; Polysciences, Inc.) as described to tracecells¹⁷. A Wilcoxon rank sum (two tailed) test is was carried out forstatistic analysis.

Subcutaneous Implantations

Subcutaneous implantations of sponges were performed in mice underanesthesia. Surgeries were conducted under sterile conditions accordingto the guidelines of the Canadian Council for Animal Care and afterapproval by the Institutional Animal Care Committee. Two subcutaneouspockets on each flank were made by a medial incision on the back of eachanimal. The FA-, TFA-, TFE-, and HFIP-treated sponges (1 cm square) wereimplanted in the same animal, one sponge in each of the four pockets.Three animals were used for each implantation time period. At 7, 15, 30and 90 days post-implantation, animals were sacrificed. Collagenspecimens were collected and fixed in formaldehyde, processed forhistological evaluation (two serial sections) and stained withhematoxylin-phloxin-saffron. Transmission electron microscopy wasperformed by drying collagen dispersion directly on grids which werethen stained with lead citrate and uranyl acetate.

Sample Processing and Agarose Gels for DNA and RNA Assays

For DNA and RNA assays, there were three groups of 5 samples: group 1included collagen and RNA, group 2 included collagen and DNA, and group3 included only collagen. For the specimens containing DNA or RNA, calfthymus DNA (Calbiochem Corp.) and yeast RNA (Boehringer Mannheim), wereintroduced during the dispersion step of collagen sponge production. DNAand RNA were used at a concentration of 0.2-0.5 μg/mg of collagen whichcorresponded to a final amount of 5 μg of DNA or RNA respectively persample. For each group, every sample was treated with the chemicalagents as described above or non-treated (control group). After chemicaltreatments, sponges were digested with a highly purified collagenase(type VII from Sigma) at 37° C. in buffer A for 2 h. A proteinase Kdigestion was performed immediately after the collagenase digestion in a1.5 ml volume. To the collagenase buffer, NaCl, EDTA pH: 7.8, proteinaseK and SDS were added to a final concentration of 85 mM, 12.5 mM, 300μg/ml and 0.5% respectively. The samples were incubated at 37° C. for 3h. After 2 h, an additional 200 μg of proteinase K per tube was added ifthe sponge was not reduced into tiny pieces. Before the threephenol-chloroform extractions, all sponges were totally digested afterthe 3 h digestion period. To precipitate the nucleic acids, 2 μl ofglycogen (20 μg/μl) and 80 μl of NaCl 5 M, 420 μl of H₂O and 4 ml ofethanol 100% were added. The nucleic acids were resuspended in 200 μl ofH₂O and transferred into a 1.5 ml microtube. Water was evaporated and 20μl of H₂O was added to resuspend the nucleic acids in a smaller volume.Five μl of 5× loading buffer [5×TAE (40 mM Tris-acetate and 1 mM EDTA,pH: 8), 0.025% bromophenol blue, 30% Ficoll 400 from Pharmacia in waterand 2% SDS] was added. The total volume (25 μl) of each sample wasloaded on a neutral agarose gel which was run at 50 volts for 2 h 10 minin TAE.

Results and Discussion

Chemical Treatment of Collagen and its Physicochemical Characteristics

Studies by FTIR spectroscopy and by DSC allow to determine respectivelythe conformational changes in collagen secondary structures and thetemperature required to reach the transition from a triple helix to arandom coil structure in collagen molecules. These are related to intra-and inter-molecular bonds.

FTIR data indicate that no strong interaction is observed betweencollagen samples and TFE or HFIP since the infrared spectra of thesesamples are almost superimposable with that of pure collagen (FIG. 1).Thus, these agents do not promote any chemical modification of collagenand are removed from the collagen sponges during the washing process. Onthe other hand, the samples treated with FA or TFA show some additionalbands, the predominant ones being located at about 1190 and 1715 cm⁻¹for FA and at 1170 and 1782 cm⁻¹ for TFA (FIG. 1). The frequencies andthe relative intensities of these new infrared peaks closely match thoseobserved in the infrared spectra of pure FA and TFA, showing that thesemolecules are still present within the collagen structure (FIG. 2). Thefrequencies of the amide I and amide II bands located near 1650 and 1550cm⁻¹ respectively, due to vibrations of the peptidic bonds of proteins,are indicative of the secondary structure adopted by these molecules. Asseen in FIG. 3, the amide I band presents a multicomponent structure dueto several factors such as amino acid composition, sequence of residuesin triplets, aggregate state of compounds, humidity of samples and typeof solvent. The main infrared feature of the amide I band of collagen iscentered at 1631 cm⁻¹ (FIGS. 3A and 3B) is characteristic of non-iminoacid residues in collagen. Although the amide I spectral region remainsunchanged for the TFE-treated samples, one can observe a modification ofthe amide I band shape for the samples treated with FA, TFA and HFIPwhen compared to untreated collagen. The deconvoluted amide I bands inFIGS. 3C and 3D evidence an additional spectral component at 1655 cm⁻¹which has been previously assigned to imino acid residues in collagen.With the exception of the TFE-treated collagen sponges, all chemicalagents lead to a decrease of the 1655/1631 absorbance ratio uponinteracting with FA, TFA and HFIP. Such a decrease in the 1655/1631ratio has been shown to be observed upon collagen denaturation. Thismodification seems to occur at the first stage of the treatment sincethe amide I region of the infrared spectra of samples treated for 1 hrare already modified with respect to that of untreated collagen. Inaddition, the relative intensity of the 1655 and 1631 cm⁻¹ features forsamples treated for 1 h are very similar to that of samples treated fora 5 h period. However, the infrared spectrum of TFA-treated sample aftera 5 h exposure period shows a considerable increase of the band width ofthe amide I feature due to the presence of spectral contribution of TFAat 1740 cm⁻¹. Thus, the amount of TFA present in collagen is higherafter an exposure for 5 h than that of 1 h. The changes in structureconformation could probably enhance trapping and grafting of FA or TFAas exposure time increases. A denaturation of collagen into gelatin maybe exhibited by an enlargement of the deconvoluted amide I feature.However, the spectra of pure gelatin (i.e. heating collagen) was foundeven larger than that after TFA (data not shown). On the other hand, theformation of FA salt with the basic groups of proteins has beendescribed and direct binding of FA at peptide bounds is also apossibility. These phenomena suggest that FA may binds to our collagen.It has been previously shown that TFA-casted polymer of poly-glycineresults in almost fully extended peptide chains, forming β-likestructure configuration in either parallel or antiparallel sheets.Similar events occur when poly-L-lysine is dissolved in TFA, withreversibility. These events could occur in TFA-treated collagen.Furthermore, perturbation of hydrogen bonds as observed with organicsolvents induces disturbances in the hydration of collagen shell whichcould affect the self-association of collagen fibrils by hydrophobiceffects. The latter may account for the destabilization of the collagenstructure.

The thermotropic transition associated with the denaturation ofuntreated collagen leads to the obtention of a large endotherm spanningfrom 45 to 80° C. centered at 61+3° C. (Table 1). This denaturationtemperature is in agreement with the value of 65° C. reported forcollagen extracted from bovine pericardia²². All chemically treatedcollagen sponges, with the exception of the one with TFE which inducedno significant modification, showed a significant decrease of thedenaturation temperature (Table 1). TFA induced the most importantdecrease of the denaturation temperature, particularly after a longperiod of exposure to chemical agents. The decrease of the denaturationtemperature observed for FA-, TFA- and HFIP-treated collagen is probablyrelated to the secondary structure modification. These agents may alsolead to a decrease of hydrogen bonds within the collagen structure. Onthe other hand, the endotherm temperature after the 5 h exposure to TFAis close to that observed with gelatin (data not shown).

Chemical Treatment of Collagen and its Biological Properties

During collagenase digestion, complete degradation of untreated collagensponges occurred within the same period (from 57 to 73 minutes averages)as treated collagen, except that HFIP-treated collagen was significantlymore rapidly degraded than was collagen treated with FA or TFE. However,the amounts of released hydroxyproline after 1 h of collagenase werefound statistically close to each other. Since collagenase targets theaminoacid sequence within the triple helix portion of collagen, thisstructure may be partially preserved after chemical treatments.

In cell culture, the determination of cell growth can reflect thepossibility of leakage of chemical agents or by-products from materials.Cell growth on treated collagen was increased as a function of time(FIG. 4), close to that seen with control sponge, except withTFE-treated sponges. In the latter conditions, cell growth wassignificantly inhibited at day 7. An explanation for this inhibitioncannot be clearly established or related to specific modifications.Conversely, it appears that eventual leakage of residual FA or TFAproducts or by-products, as shown by FTIR, has not targeted cells inculture.

Untreated collagen materials prior to implantation appeared as periodiccollagen fibril bundles forming a porous structure¹⁴⁻²³. Similarstructures were observed in TFE- or HFIP-treated collagen. In opposite,collagen fibril bundles were closely packed (FIG. 5A), resembling acollapsed pore structure in FA- or TFA-treated samples. These fibrilsremained periodic following FA treatment (FIG. 5B), but not with TFA, asobserved by transmission electron microscopy. The latter observation(i.e., TFA treatment) suggests that denaturation occurred in each fibrilwithout entirely melting the whole collagen material. Conversely,denaturation by FA treatment may be limited to some collagen fibrils.

Studies of in vivo behaviour of these materials allows to investigatetheir biological properties within a complex cell and tissueenvironment. After implantation for 7 days, cell infiltration consistedof few inflammatory cells and fibroblasts within the collagen implants.Inflammatory reaction was also present in the tissue surrounding theimplant, particularly in sponges treated by TFA and HFIP (Table 2). By15 days, infiltration by inflammatory cells and fibroblasts was presentwithin the implants treated particularly with FA, TFE and HFIP.Adipocytes or fatty degenerescence was observed within collagenmaterials treated by TFE. Inflammation was especially noticeable in theperiphery of TFA-treated collagen. By 1 month, cell infiltrationoccurred in all chemically-treated sponges (FIG. 5). With FA-, HFIP-,and TFE-treated collagen, cell infiltration was present within the wholeinterior of collagen materials and in addition, new connective tissueappeared at various sites between the implanted collagen bundles. WithTFA-treated collagen samples, the implant appeared partially resorbedwith fatty tissue accumulation and persistence of an inflammatoryreaction. By 90 days, implants had been largely resorbed, although someresidual collagen was observed, except with HFIP-treated collagen forwhich no implant was retrieved. After FA and TFA treatments whichexhibit significant decrease in denaturation temperature, the residualcollagen was surrounded by a slight inflammatory reaction andinfiltrated by newly deposited collagen. The inflammatory reaction wasminimal in the periphery and the interior of FA-treated implantscompared to other treatments. In addition, the response lastedapparently for less than 30 days. These responses were also observedwith sponges treated by TFE. In opposite, TFA-treated sponges induced animportant inflammatory response that could be explained by the presenceof a high residue of TFA within collagen compared to those treated byFA. Inflammatory reaction to HFIP-treated sponges was apparentlyimportant, but limited to the early period of implantation.

In comparison to previous studies²³⁻²⁴ using collagen sponges as a woundscaffold, the implantation of a collapsed porous structure resulting ofFA and TFA treatments induces a rapid cell infiltration. Similarphenomena have been described with denaturated collagen implants orafter blending collagen fibrils with gelatin^(25,26). Denaturated andnon-denaturated collagen may be present in our material as shown by FTIRand DSC. Collagen in acids swells with alteration of fibril lengths andthicknesses, while the triple chain units remain intact²⁷. With FA orTFA treatment, it is less probable that under acid treatment collagenconverts totally to gelatin since our starting collagen consists ofrigid fiber units that can slow the denaturation process through theresistance force of these fibers. However, the treatment by TFA, morespecifically after a 5 h exposure, probably induces more dissociation ofcollagen fibrils than with FA, as demonstrated with denaturating agentsand by our transmission electron microscopic observation. Sinceincreasing potential of denaturation occurred after long exposure tochemical agents, the 1 h exposed specimens have been investigatedtowards cell culture and in vivo biocompatibility.

Effects of Chemical Treatments on Nucleic Acids in Collagen

Analysis of single-strand RNA fragment and double-stranded DNA fragmentmobility distribution on agarose gels is a useful method for determiningthe frequency of strand breaks which is correlated with the degree ofnucleic acid degradation (reviewed in Drouin et al.,²⁹). Neutral agarosegel can visualizes as little as 2 ng in a 0.5-cm-wide band³⁰. Thesensitivity is 5- to 10-fold lower for single-stranded DNA and RNA thanfor double-stranded DNA. This means that about 20 ng of single-strandednucleic acids are needed to be easily detected. The collagen obtainedfrom bovine hide does contain some nucleic acids (FIG. 7, lanes in C).These nucleic acids are likely to be mainly RNA with an average fragmentlength inferior to 100 nucleotides. The upper part of the smear isprobably aggregated ribosomal RNA and tRNA. DNA likely represents asmall part of the nucleic acids present in the bovine hide extracts. Thepresence of minidose of nucleic acids could be due to (i) bacteria asdemonstrated by microbiological analysis (environmental bacillus andmycobacterium: “data not shown”); (ii) viruses (non-demonstrated),and/or (iii) residues of cells remaining after hair removal from bovineskin at the tannery, and/or after purification by acetic acid dispersionand salt precipitation. No cell nucleus was found to be present inobservations of histological sections of freeze-dried purified collagenand virgin collagen sponges using specific DNA dye (“data not shown”).

On the other hand, TFA hydrolyzed any nucleic acids to very smallfragments (less than 10 nucleotides); even the added DNA and RNA weretotally degraded (lanes A3 and B3, FIG. 6). FA also hydrolyzed anynucleic acids contained in the collagen sponges to very small fragmentswhich were not recovered, whereas the added RNA and DNA were degraded toan average fragment length of less than 40 nucleotides. The otherchemical treatments did not seem to induce any significant amount ofstrand breaks (lanes A4, B4 and C4, FIG. 6). In all likelihood, the veryacidic conditions of the FA (pH: 1) and TFA (pH: 1) treatments causedextensive DNA denaturation, depurination of the nucleic acids, andhydrolysis of the phosphodiester bonds of both RNA and DNA. Conversely,treatment of collagen by 1N NaOH, as recommended by most companiesproducing collagen, does not break down DNA or RNA as recentlydemonstrated by neutral gel agarose (data not shown).

Every chemical treatment tested in this work involves either a strong(pH: 1) or a mild (pH: 4.5-5) acidic environment, and treatments wereperformed at room temperature for at least 1 h. Under the strong acidicconditions, there are denaturation of the DNA, an extensive depurinationof both DNA and RNA, and hydrolysis of the phosphodiester bonds of bothpolydeoxyribonucleotides and polyribonucleotides³¹. The purine residuesare readily removed from DNA by mild acid treatment. In mild acidicconditions, DNA is partially denaturated and partially depurinated; RNAis basically untouched and very few phosphodiester bonds are hydrolyzedin both RNA and DNA. In summary, strong acid treatment of collagensponges definitively leaves DNA, too depurinated, and RNA and DNA withtoo many strand breaks to be usable by any DNA or RNA polymerases to beinfectious; whereas mild acidic treatment leaves RNA molecules which canbe easily copied to synthesize DNA while the degree of depurination ofthe DNA might be so important that its infectivity will be precluded.Furthermore, treatment by a chemical scrapie inactivator (e.g., FA orTFA) induced the loss of the β sheet-like secondary and tertiarystructure of prion that correlates with inactivation of scrapieinfectivity¹¹. Based on the results of the present study concerning thedegradation of nucleic acids by FA and TFA treatments and thosepreviously reported¹¹, we can conclude that the chemically-treatedcollagen lacks both scrapie infectivity and viral transmission. Inconclusion, such a chemical treatment could constitute a method toproduce safe collagen/gelatin materials protected against prions andviruses. FA seems to be the most efficient of the four tested agentsbecause of its activity as a chemical inactivator in degrading nucleicacids, and its use results in a good biocompatible collagen/gelatinmaterial, despite its presence within the collagen molecule. The latterinduces only a temporary inflammatory response, with minimalinflammation as observed with other biodegradable and non-biodegradablebiomaterials.

From the above results, it can be deduced that the two acids whichachieved elimination of prion are strong organic acids having a pH ofabout 1. The two other compounds having a pH of about 5.0 were notefficient. Other strong organic and/or inorganic acids may be equivalentto TFA and FA provided that THEY achieve a solution pH below about 2.0;and provided that they do not degrade collagen to an undesirable extent.The time of reaction may be adjusted in function of the acidic strengthof the acid agent.

Dehydrothermal Treatment:

As mentioned above, a plurality of known sterilizing procedures havebeen tried for the production of safe collagen products (e.g. NaOH,glutaraldehyde, urea). From the recommended use of NaOH as a treatmentof collagen, one can deduce that a heat treatment is to be avoided. Wehowever tried to sterilize collagen, using milder conditions thanautoclaving at 134° C. Collagen was dehydrated under vacuum for 1-3 daysat 110° C. in an oven. Collagen was slightly crosslinked and sterileupon treatment, without denaturation. In these conditions, we haveanalyzed, by agarose gel, DNA and RNA degradation, and observed acomplete break down of these molecules (using DNA and RNA added tocollagen). The time of dehydrothermal treatment may vary for 1 to 3 dayswhen the temperature is fixed to 110° C., can be more or less shortenedat higher temperatures or lengthened at lower temperatures.

Since autoclaving is not desirable for maintaining collagen integrity,it is therefore apparent that there is a limit temperature and pressurevalue beyond which collagen is destroyed in an unacceptable proportion,and this is observed either in wet conditions as occurred in autoclaveor in dry heating under atmospheric humidity. If any water vapour orhumidity is not completely removed (for example using a high vacuum)prior to increasing temperature, collagen will begin to denature andthen be destroyed sooner than viruses, bacteria or prions.

Thermal treatment may be applied directly to gelatin, and will have as adouble advantage to stabilize gelatin by crosslinking and to produce asafe prion-free product. If gelatin is prepared by treating collagenwith a strong acid like TFA for a prolonged time (more than about 5days), heat treatment would provide a double safe product (TFA and heatare two process steps eliminating prion) and will also stabilize gelatin(which is an altered collagen). Crosslinked gelatin could be used toimplement enteral resistance of a gelatin capsule for drug delivery, forexample.

Properties of Collagen Sponges Treated With TFA as Drug DeliverySystems:

(i) The treatment of collagen with TFA (1 hr exposure) induced highwater adsorption and absorption (see FIG. 7) while FA treatment appearedto impair water sorption. This has been also observed with theabsorption of peptides such as growth factors. Our investigation showedthat radiolabeled growth factor is uniformly distributed within thecollagen porous structure treated with TFA while after FA treatment thedistribution of radiolabeled growth factor remained around the sponge asdetermined by autoradiography. TFA property is very interesting toachieve the design of drug absorption onto collagen materials such asthat currently described.

(ii) A stable porous structure of collagen sponge, free of prions andviruses can be used as a wound dressing or drug delivery system.Collagen materials which have been stabilized by polyethylene glycol(PEG) as described in the patent publication CA 2,164,262 (PEG-collagen)can be treated by FA or TFA. However, the treatment with FA or TFAshould be performed after PEG grafting in order to preserve the porousstructure. These composite products have been investigated towards theirbiocompatibility. They offer similar behaviour than that described foruntreated PEG-collagen. Results in cell culture show no cytotoxicity ofthe products (cell growth was similar to that of the control), andanimal studies (mice) have shown slight inflammatory reaction with cellinfiltration. The porous structure remained stable for 180 days in mice,except for PEG-collagen treated by TFA. The latter lasted for 90 days ofimplantation, biodegradation occurred then. This variety of stabilitycan be beneficial to offer various products with a range ofbiodegradation rates. A PEG-collagen treated with TFA could be furthersubmitted to severe dehydrothermal treatment, which promotescrosslinking and increases stability. We have verified whetherPEG-grafted to collagen impairs DNA and RNA destruction by FA or TFAtreatment. Gel agarose showed clearly the breakdown of nucleic acids asreported previously on collagen sponges.

(iii) When FA and TFA are introduced before the PEG treatment, collagensponges failed to remain porous. However, resulting collagen productsallowed cell infiltration, with some delay during the wound healingprocess. This interesting property can be used to stabilize capsulesmade of gelatin (such as induced by TFA) as mentioned above.

(iv) The composite PEG-collagen can be useful as a growth factordelivery system, because it can maintain higher concentrations of growthfactor, compared to collagen alone. The treatment of this compositePEG-collagen by FA or TFA preserved in vivo this property (see FIG. 8).In addition, by autoradiography, the growth factor is well distributedwithin the porous structure.

Transparent Products and Implants

Transparent or clear collagen is obtained by prolonged exposure to TFAwhich can vary from 6 to 12 hours, depending on the collagen batch.Thus, treated collagen sponges behave as hydrogel-like materials with atransparency property. Treated collagen film (air-dried dispersion) ismore fragile than treated collagen sponges (or hydrogels).

Transparent collagen materials can be produced in various thicknessesranging from 10 to 500 μm. The concentration of collagen dispersion isalso an important parameter in getting transparency. Thus, a 0.5 to0.75% (weight of collagen to volume of water) appeared transparent. At1% collagen dispersion, transparency was never reached.

On the other hand, the addition of hyaluronic acid (HA), aglycosaminoglycan, enhances the transparency of the collagen (5% (w/w)HA per weight collagen, HA being added to the collagen dispersion). Thishas been determined by measuring optic density at different wavelengthadsorption (see FIG. 9). The addition of other glycosaminoglycans andproteoglycans and other percentages of HA can be also beneficial ingetting a more transparent material. Furthermore, the addition ofhyaluronic acid stimulates cell infiltration into the implant asdemonstrated in cell culture.

Those transparent or clear materials can be used for ocularapplications, and more particularly for transparent corneal wounddressings. Collagen shields are used for the same purpose, however, theyare produced from bovine, swine, or human, but their safety remained anissue. Our product could safely be used.

This invention has been described hereinabove and it will become readilyapparent to the skilled reader that modifications can be made theretowithout departing from the above teachings. These modifications areunder the scope of the invention as defined in the appended claims.

REFERENCES

1. E. Bell, B. Ivarsson, and C. Merril, “Production of a tissue-likestructure by contraction of collagen lattices by human fibroblasts ofdifferent proliferative potential in vitro,” Proc. Natl. Aca Sci. USA,76, 1274-1278 (1979).

2. I. V. Yannas, J. F. Burke, D. P. Orgill, and E. M. Skrabut, “Woundtissue can utilize a polymeric template to synthesize a functionalextension of skin,” Science, 215, 174-176 (1982).

3. T. Miyata, T. Taira, and Y. Noishiki, “Collagen engineering forbiomaterial use,” Clin. Mater. 9, 139-148 (1994).

4. G. Ellender, R. Papli, R. Hammond, K. Mitrangas, J. F. Bateman, V.Glattauer, J. M. Thyer, J. A. Werkmeister, and J. A. M. Ramshaw,“Osteogenic capacity of collagen in repair of established periodontaldefects,” Clin. Mater. 9, 201-209 (1994).

5. J. Keefe, L. Wauk, S. Chu, and F. DeLustro, “Clinical use ofinjectable bovine collagen: A decade of experience,” Clin. Mater., 9,155-162 (1994).

6. S. B. Prusiner, “Novel proteinaceous infectious particles causescrapie,” Science, 216, 136-144 (1982).

7. S. B. Prusiner, “Inherited prion diseases,” Proc. Natl. A cad. Sci.USA, 91, 4611-4614 (1994).

8. P. Brown, P. P. Liberski, A. Wolff, and D. C. Gajdusek, “Resistanceof scrapie infectivity to steam autoclaving after formaldehyde fixationand limited survival after ashing at 360 degrees C: Practical andtheoretical implications,” J.Infect. Dis., 161, 467-472 (1990).

9. R. N. Rosenberg, C. L. White, P. Brown, D. C. Gajdusek, J. J. Volpe,J. Posner, and P. J. Dyck, “Precautions in handling tissues, fluids andother contaminated material from patients with documented or suspectedCreutzfeldt-Jakob disease,” Ann. Neurol., 19, 75-77 (1986)

10. J. Tateichi, T. Tashima, and T. Kitamoto, “Inactivation ofCreutzefelt-Jakob disease agent,” Ann. Neurol., 24, 466- (1988).

11. J. Safar, P. P. Roller, D. C. Gaidusek, and C. J. Gibbs, “Thermalstability and conformational transitions of scrapie amyloid (prior)protein correlate with the infectivity,” Protein Sci., 2, 2206-2216(1993).

12. W. E. Klunk, C.-J. Xu, and J. W. Pettegrew, “NMR identification ofthe formic acid modified residue in Alzheimer's amyloid protein.” J.Neurochem., 62, 349-354 (1994).

13. E. J. Miller, and R. K. Rhodes, “Preparation and characterization ofthe different types of collagen,” Methods Enzymol., 82, 33-64 (1982).

14. M.-F. Côté, E. Sirois, and C. J. Doillon, “In vitro contraction rateof collagen in sponge shape matrices,” J. Biomater. Sci. -Polym. Ed., 3,301-313 (1992).

15. P. R. Griffiths, and G. L. Pariente, “Introduction to spectraldeconvolution,” Trends Anal. Chem., 5, 209-215 (1986).

16. R. A. Berg, “Determination of 3- and 4-hydroxyproline,” MethodsEnzymol., 82, 372-398 (1992).

17. J. L. Ellwart, and P. Domer, “Vitality measurement using spectrumshift in Hoechst 33342 stained cells.” Cytometry, 11, 239-243 (1990).

18. Y. A. Lazarev, B. A. Grishkovskii, and T. B. Khromova, “Amide I bandof IR spectrum and structure of collagen and related polypeptides.”Biopolymers, 27, 1449-1678 (1985).

19. K. Payne, A. Veis, “Fourier transform IR spectroscopy of collagenand gelatin solutions:

deconvolution of the amide I band for conformational studies.”Biopolymers, 27, 1749-1760 (1988).

20. A. Veis, The Macromolecular Chemistry of Gelatin, Molecular Biology,volume 5, Horecker, B. Kaplan, N. O. & Scheraga H. A. (eds.), AcademicPress, New York-London, (1964).

21. A. V. Kajava, “Molecular packing in type I collagen fibrils. A modelwith neighbouring collagen molecules aligned in axial register.” J. Mol.Biol., 218, 815-823 (1991).

22. J. M. Lee, C. A. Pereira, D. Abdulla, W. A. Naimark, and I.Crawford, “A multi-sample denaturation temperature tester forcollagenous biomaterials,” Med. Eng Phys., 17, 115-121 (1995).

23. C. DeBlois, M.-F. Côté, and C. J. Doillon, “Heparin-FGF-fibrincomplex: In vitro and in vivo applications to collagen-based materials,”Biomaterials, 15, 665-672 (1994).

24. C. J. Doillon, M. G. Dunn, R. A. Berg, and F. H. Silver, “Collagendeposition during wound repair,” Scan. Microsc., 2, 897-903 (1985).

25. K. Yoshizato, and E. Yoshikawa, “Development of bilayered gelatinsubstrate for bioskin: a new structural framework of the skin composedof porous dermal matrix and thin basement membrane,” Mater. Sci. Eng.C-Biomimetic Materials, Sensors & Systems, 1, 95-105, (1994).

26. M. Koide, K. Osaki, J. Konishi, K. Oyamada, T. Katakura, A.Takahashi, and K. Yoshizato, “A new type of biomaterial for artificialskin: dehydrothermally cross-linked composites of fibrillar anddenaturated collagens,” J. Biome. Mater. Res., 27, 79-87 (1993).

27. GN Ramachandran, in Collagen, Ramanathan N. (ed.), WileyInterscience, New York, (1962).

28. J. H. Lillie, D. K. MacCallum, L. J. Scaletta, and J. C. Occhino,“Collagen structure: Evidence for a helical organization of the collagenfibril,” J. Ultrastruct. Res., 58, 134-143 (1977).

29. R. Drouin, S. Gao, G. P. Holmquist, “Agarose gel electrophoresis forDNA damage analysis”. In: Technologies for detection of DNA damage andmutations. G. P. Pfeifer (ed), Plenum Press, New York, 1996, (in press).

30. J. Sambrook, E. F. Fritsch, and T. Maniatis, eds. Molecular Cloning:A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

31. D. M. Brown, “Chemical reactions of polynucleosides and nucleicacids” in Basics Principles in Nucleic Acid Chemistry Vol II, Ts'O PaulOP ed. Academic Press, New York-London, pp.1-90 (1974).

TABLE 1 Denaturation temperature for the untreated and chemicallytreated collagen sponges A B Collagen Denaturation temperature (± 3° C.)untreated 61 61 FA 41 36 TFA 35 29 TFE 57 58 HFIP 43 39

Collagen sponges where chemically treated with FA, TFA, TFE or HFIP for1 h (A) and 5 h (B) exposure periods.

TABLE 2 Qualitative scale of the inflammatory reactions afterimplantation of chemically-treated collagen sponges Tri- Formicfluoroacetic Trifluoro Hexafluoro- acid acid ethanol 2-propanol Days P IP I P I P I  7 ± ± ++ ± ± ± ++ + 15 ± ± ++ + + + ± + 30 + 0 + + ± 0 ± ±90 ± 0 ± 0 0 0 Not retrieved The inflammatory reaction was qualitativelyappreciated following a relative scale: ++ for extensive; + formoderate; ± for light; and 0 for nil to sparsely light. Reactions werenoted in the periphery (P) and inside (I) of the implants.

We claim:
 1. A process for producing a compound comprising collagen freeof prion, comprising: a) treating said compound with an organic acidhaving a solution pH equal to or below 2 for a period of time of atleast one hour, wherein said prion is eliminated while said compoundcomprising collagen is not denatured after said treatment process, whileat least a part of said collagen is converted into gelatin.
 2. A processas defined in claim 1, wherein said acid is a pure undiluted organicacid.
 3. A process according to claim 1, wherein said period of time isabout one hour.
 4. A process according to claim 1, wherein said compoundcomprising collagen is obtained from a solution comprising about 0.5 toabout 0.75% (w/v) collagen, and wherein said compound comprisingcollagen obtained at the end of the organic acid treatment step istransparent.
 5. A process according to claim 1, wherein said compoundcomprising collagen is a polyethylene glycol-grafted collagen which isformed as a porous material.
 6. The process of claim 1, further whereinand infectious agents are eliminated.
 7. A process according to claim 1,wherein said organic acid is trifluoroacetic acid or formic acid.
 8. Aprocess according to claim 7, wherein said organic acid is pure formicacid.
 9. A process according to claim 7, wherein said organic acid ispure trifluoroacetic acid.
 10. A process as defined in claim 1, whereinsaid period of time is at least five hours, whereby the prion iseliminated while at least a part of collagen is converted into gelatin.11. A process according to claim 10, which further comprises the step ofcrosslinking the gelatin after the organic acid treatment step.
 12. Aprocess according to claim 11, wherein the crosslinking is achieved byan organic aldehyde or dehydrothermal treatment.
 13. A process accordingto claim 12, wherein the dehydrothermal treatment comprises heating atabout 110° C. for about 1 to 3 days under high vacuum.
 14. A process forproducing a compound comprising collagen which is free of prion whichcomprises treating the compound comprising collagen to a temperature ofabout 110° C. for a period of time and conditions sufficient toeliminate prion while said compound comprising collagen is not denaturedafter said treatment process, while at least a part of said collagen isconverted into gelatin.
 15. A process according to claim 14, whereinsaid period of time is comprised between about 1 day to about 3 daysunder high vacuum.